Vessel for blood sampling

ABSTRACT

The present invention relates to a vessel for withdrawing blood, the vessel containing a solution which comprises a guanidinium salt, a buffer substance, a reducing agent, and/or a detergent as components. The vessel is particularly suited for withdrawing blood which is to be analyzed with respect to nucleic acids.

CROSS REFERENCE TO RELATED APPLICATIONS

Applicant claims priority under 35 U.S.C. §119 of GERMAN Application No.198 36 559.4 filed on Aug. 12, 1998. Applicant also claims priorityunder 35 U.S.C. §120 of PCT/EP99/05857, filed on Aug. 12, 1999. Theinternational application under PCT article 21(2) was not published inEnglish.

The present invention relates to a vessel for withdrawing blood, and theblood withdrawn should especially be used for stabilizing and analyzingnucleic acids.

When blood is taken, it is normally collected in vessels which alreadycontain anticoagulants such as heparin, citrate or EDTA. The blood isthereby prevented from coagulating. The blood samples obtained therebycan be stored at suitable temperatures for a long time. This way ofobtaining blood has, however, considerable drawbacks when nucleic acidssuch as (m)RNA or DNA are to be analyzed. For such purposes the nucleicacids contained in the sample should optimally be stabilized already atthe moment of withdrawal, i.e. a degradation of the existing nucleicacids should be prevented, but also the new synthesis of mRNA.

This objective of a stable storage of the nucleic acids contained in thesample material, i.e. from the moment of withdrawal, has not beenachieved yet in practice for the following reasons:

Cells contain nucleases, enzymes, which destroy nucleic acids as soon asthey come into contact with the substrates thereof (RNA, DNA). Theeffect of cellular and extracellular nucleases is normally underphysiological control as long as the cells are in their normalenvironment. The withdrawal of blood effects more or less strong changesin the nucleic acids contained in the cells. Nucleases are then releasedwithin the cells and/or by the lysis of cells to the outside. Moreover,nucleic acids are synthetized more or less strongly. In particular thelong-term storage of blood leads to aging and destruction of the cells.

Another problem arising in the long-term storage of blood samplesobtained according to standard withdrawal methods is the considerablechange in the sample material. Such changes, e.g. strong lysis of cells,may have the effect that the standard methods for isolating nucleicacids no longer function in an adequately efficient way.

Apart from the problems regarding a stable storage of nucleic acidscontained in the sample material, further difficulties arise in theconventional method for withdrawing blood. The conventionalanticoagulants are often not separated efficiently enough duringisolation of nucleic acids and interfere with in the subsequent analysisof nucleic acids, e.g. in the case of amplification by means of PCR(polymerase chain reaction). Heparin is e.g. a generally known inhibitorof PCR.

Finally, the question arises in the quantitative analysis of nucleicacids how the whole method ranging from sampling to the measurement ofnucleic acids can be controlled under standardized conditions. Ideally,a quantitatively and qualitatively defined standard nucleic acid shouldalready be added to the sample material during withdrawal and should besubjected to the whole process of sampling and determination. This canalso not be accomplished with the conventional withdrawal systems.

A further drawback of conventional blood withdrawal is the risk oftransferring infectious material because manual process steps have sofar been needed for the isolation of nucleic acids. Contact withpotentially infectious germs cannot be ruled out.

In the literature there is described a method in which the blood sampleis mixed with guanidinium salt directly after withdrawal from a patient(EP 0 818 542 A1). In this method the guanidinium salt is present inpowder form to thereby exploit the increased stability of theguanidinium salt. This method, however, has serious drawbacks becausethe salt, for instance, must first dissolve in the added blood. Thisdissolution process depends, in particular, on the temperature andcannot be controlled because of the nontransparent sample material used.The use of a corresponding product for diagnostic medical purposes isthus very problematic.

Furthermore, nucleases are extremely active enzymes which can only beinhibited under extremely denaturing conditions. Denaturation depends onthe concentration of the guanidinium salt in solution. An inhibitingconcentration of guanidinium salt in solution does not exist in thecited method right from the beginning. Thus, there is an uncontrolleddegradation of nucleic acids during the dissolution process. Moreover,in this method the addition of reducing agents is omitted, without whichan efficient inhibition, in particular of RNases, is not ensured (seeExample no. 5).

Moreover, the sample prepared in this way cannot directly be used forthe further nucleic acid isolation on glass surfaces. Moreover, the useof guanidinium salt powder does not permit the addition of internalnucleic acid standards. Such standards are mandatory for process controland exact quantification.

The present invention has been based on the technical problem ofproviding a vessel for withdrawing blood which does not have thedrawbacks of the prior art. In particular, it should be possible tosubject the sample taken with the vessel directly to the standardmethods for analyzing nucleic acids without the need for further samplepreparation steps.

According to the invention this problem is solved by a vessel forwithdrawing blood, the vessel containing an aqueous solution comprisingthe following components:

-   -   a guanidinium salt;    -   a buffer substance;    -   a reducing agent; and/or    -   a detergent.

The vessel of the invention has the following advantages: 1. Blood isalready lysed at the moment of withdrawal in that the withdrawal vesselalready contains a nucleic acid-stabilizing substance in solution. 2.The nucleic acid-stabilizing substance is composed such that the samplematerial, in particular the nucleic acids contained therein, aredirectly stabilized upon contact with the solution. 3. In contrast toall of the former standard withdrawal systems, such as EDTA orheparin-containing withdrawal vessels, the stabilized sample need nolonger be handled as infectious material. 4. The nucleicacid-stabilizing substance is composed such that the sample material candirectly be used in subsequent isolating methods. 5. The nucleicacid-stabilizing substance can be separated during subsequent isolationso efficiently that an inhibition of PCR is not observed. 6. The nucleicacid-stabilizing substance may have added thereto an internal standard.This permits the control of the whole method from the moment of samplingup to the detection of nucleic acids.

The withdrawal vessel mentioned under item 1 is a conventional bloodwithdrawing vessel (small tube) which has introduced thereinto a definedvolume of a nucleic acid-stabilizing substance. The small tube is thenpreferably subjected to a defined vacuum which guarantees that only aspecific volume of blood can flow thereinto during withdrawal. The smalltube can be handled by conventional blood-taking methods. The solutioncontained in the tube contains the following reagents in a speciallypreferred embodiment: Guanidinium thiocyanate, Triton-X-100,dithiothreitol and a suitable buffer system, such as citrate, Tris orHEPES. In the described composition the solution is compatible with thevacuum tube. This solution can be stored in the vacuum tube without anyproblems and without any impairment of the desired stabilizing function.The whole system presents no problems, in particular to blood donors,and is safe during sampling.

The solution containing the guanidinium salt, the buffer substance, thereducing agent and/or the detergent is stable in storage and convertsthe supplied and freshly taken blood into a material which is alsostable in storage and can directly be subjected to the standardnucleic-acid analysis kits (e.g. those of Roche or Qiagen).

Guanidinium thiocyanate and/or guanidinium chloride are preferred asguanidinium salt.

Preferably, the guanidinium salt is present in a concentration of 2.0 to8.0 M. Tris or citrate is preferred as the buffer substance, the exactpH being preferably adjusted with HCl. Further possible buffers arehowever HEPES, MOPS, citrate and phosphate buffer, such as PBS.

The buffer concentration is preferably between 10 and 300 mM,particularly preferably between 10 and 100 mM.

Triton-X-100 is preferred as the detergent. Further possible detergentsare NP40, Tween 20, polydocanol or other detergents.

The detergent concentration is preferably at 5 to 30% (w/v),particularly preferably at 10 to 20% (w/v).

DTT is preferred as the reducing agent, but β-mercaptoethanol, TCEP(Tris(2-carboxyethyl)phosphine) or other reducing agents can also beused.

The preferred concentration of the reducing agent is at 0.1 to 10%(w/v), particularly preferred are 0.5 to 2% (w/v).

The pH of the solution is preferably at 3.0 to 9.0, particularlypreferably at 4.0 to 7.5, particularly preferably at 5 to 6.

The pH of the solution is in particular chosen such that a pH rangingfrom 5.0 to 7.6 is set after addition of the sample material.Particularly preferred is a pH between 6.3 and 6.9 (see Example no. 8).

A particularly preferred solution preferably contains 4 M guanidiniumthiocyanate, 45 mM Tris/HCl, 18%, preferably 15% (w/v) Triton-X-100,0.8% (w/v) DTT and has a pH of 6.0.

In a further preferred embodiment the volume for receiving the bloodsample has a negative pressure which can be adjusted such that apreviously determined blood volume is sucked into the vessel after ablood vessel has been pierced. Correspondingly evacuated vessels areavailable on the market.

The vessel which contains the blood taken can then immediately besubjected to further analyses or, however, may be stored for a longperiod of time (up to several days) without any disadvantages for thequality of the sample.

In the method of the invention the freshly taken blood is directlycontacted in the blood withdrawing vessel with the above-describedsolution so that all processes which might change the nucleic acidpattern of the sample are immediately stopped. Therefore, the datadetermined at a later time with respect to the detected nucleic acidsvery accurately represent the actual state at the time of bloodwithdrawal, i.e. both with respect to the quantities and the types ofnucleic acids.

Preferably, the blood amount taken is 0.1 to 4 times the solution fedinto the vessel. The solution is preferably 0.5 to 5.0 ml. Thus thefinal concentration of guanidinium salt after blood addition is at 1.0to 5 M, preferably at 1 to 3 M, particularly preferred are 2-3 M (seeExample 7).

The vessel according to the invention is preferably used for bloodwithdrawal when the blood sample is to be used for analyzing nucleicacids. The use of the above-mentioned solution as a component of thedescribed withdrawal system solely guarantees the immediate lysis of thecells and the simultaneous stabilization of the sample by immediateinactivation of the nucleases. Surprisingly, the blood sample obtainedthereby can be stored even at room temperature or higher for severaldays. Moreover, the withdrawal system guarantees a contamination-freeand non-infectious handling ranging from sampling via nucleic acidisolation to analysis. In the conventional methods of nucleic acidisolation, additional handling steps have so far been required (e.g. thetransfer of the blood sample taken into the reagents for nucleic acidisolation, etc.), which entails an additional risk of infection.

The sample obtained with the blood withdrawing system is compatible withall of the conventional standard methods of nucleic acid isolation.Particular attention should here be paid to methods which are based onthe binding of nucleic acids to glass surfaces, but alsosequence-specific binding to complementary nucleic acid andsolvent-based extraction methods.

Thus the invention as described consists of a blood withdrawing systemwhich is conceived such that the following conditions are satisfied. 1.Controlled sampling and simultaneous stabilization of the nucleic acids(DNA, RNA) contained in the sample material. 2. Sampling in which theuse of anticoagulants can be completely omitted. 3. The sample obtainedby way of the above-described blood withdrawing system can be used in auniversal manner in all of the known systems for isolating nucleicacids. 4. The blood withdrawing system is stable in storage.

Additionally, it has surprisingly been found that the sample obtained byway of the described withdrawal system can be stored in the vessel for along period of time without degradation of the nucleic acids (seeExamples 2, 3, 7, 8).

The following examples will explain the invention:

EXAMPLE 1

The blood withdrawing system may be composed in a preferred embodimentas follows (see FIG. 1): A small tube is filled with a defined volume ofthe nucleic acid-stabilizing substance and is provided with a definedvacuum and sealed with a septum. The septum is constructed such that itis compatible with the standard sampling accessories (cannula, etc.). Inthe present example 2.2 ml reagent was supplied and the vacuum wasadjusted such that exactly 2.2 ml blood could flow in during sampling.The nucleic acids contained in the inflowing blood flow were immediatelyconverted into a stable form.

General preliminary remark regarding the following examples.

In all of the examples described hereinbelow, the nucleicacid-stabilizing substance (N-sS) had, unless indicated otherwise, thefollowing composition: 45 mM Tris, 5 M guanidinium thiocyanate (GTC),0.8% (w/v) dithiothreitol (DTT), 18% (w/v) Triton-X-100, pH 6.0.

In all of the examples described, the nucleic acid-stabilizing substancewas, unless indicated otherwise, mixed with the sample in the ratio of 1to 1 (1 volume N-sS plus 1 volume sample material). A lowerconcentration of N-sS, e.g. 1 volume N-sS plus 5 volumes sample, mighteffect a degradation of RNA.

Blood was stabilized for all examples by directly feeding the blood uponwithdrawal into the small tube mixed with N-sS.

EXAMPLE 2

Stability of nucleic acid after mixture of sample material and N-sS.Isolation of RNA and DNA from the sample lysate with silica-derivatizedsurfaces.

Material and Method

The sample material for the DNA and RNA isolation was directly usedafter withdrawal, after storage at 4° C. for 6 days, and after storageat −20° C. for 1 month.

The High Pure RNA Isolation Kit (Boehringer Mannheim, cat. no. 1828 665)was used for isolating RNA (FIG. 2). The instructions given in thepackage leaflet were modified as follows: A volume of 2.4 ml samplelysate was applied in 4 aliquots at 600 μl each to the column, so that asample material of 2.4 ml lysate was applied on the whole. All of theother steps were carried out in accordance with the package leaflet. TheRNA was finally eluted with 100 μl elution buffer.

For the isolation of DNA (FIG. 3) the QiaAmp Blood Kit (Qiagen cat. no.29104) was used. The standard procedure described in the package leafletwas modified in various points: 400 μl sample volume was directlyapplied to the column; the binding reagent contained in the kit was notused. 25 μl proteinase-K batch solution was added and the sample wasincubated at room temperature for 10 min. Subsequently, the column wasput into a collection vessel and centrifuged as described in the packageleaflet. All of the further steps were carried out in accordance withthe description in the package leaflet, except for the use of ethanol.The elution volume was 200 μl.

EXAMPLE 3

Isolation of mRNA from sample lysate using streptavidin-coated magneticparticles and biotin-labeled Oligo(dT) (FIG. 4):

Material and Method

20 ml sample lysate was fed into a vessel. The mRNA was isolatedaccording to the following method: First of all 30 ml hybridizationbuffer (20 mM Tris-HCl, 300 mM NaCl, 6 nM biotin-labeled Oligo(dT), pH7.4) was added to the lysate. 3 mg streptavidin magnetic particles(Boehringer Mannheim) were then added. The sample was mixed andincubated at room temperature for 5 min. The magnetic particles wereseparated with the help of a magnet; the supernatant was discarded. Theparticles were then resuspended in wash buffer 1 (10 mM Tris-HCl, 200 mMNaCl, 1% Triton-X-100, pH 7.5) and washed three times with wash buffer 2(10 mM Tris-HCl, 200 mM NaCl, pH 7.5) (wash steps: resuspension,magnetic separation, removal of the supernatant). After the last washstep the supernatant was completely removed and the particles wereresuspended in 20 μl distilled water. The sample was heated to 70° C.for 5 min. The magnetic particles were separated, and the supernatantwhich contained the mRNA was analyzed by means of gel electrophoresis.

EXAMPLE 4

Isolation of DNA and RNA using a modified rule according to Chomczynskiand Sacchi (Analytical Biochemistry 162, 156-159 (1987)) (example of amethod based on solvent extraction) (FIG. 5):

Material and Method

2 ml sample volume was transferred from the blood withdrawing vesselinto a small tube. 0.2 ml of a 2 M sodium acetate solution, pH 4, 2 mlphenol (water saturated) and 0.4 ml of a chloroform-isoamyl alcoholmixture (49:1) were then added, the sample being thoroughly mixed afteraddition of each solution. The complete solution was vigorously shakenfor 10 seconds and incubated on ice for 15 minutes. The sample wascentrifuged for 20 minutes at 4° C. at 10000 g. After centrifugation theRNA was in the aqueous phase; the DNA and proteins in the intermediateand phenol phase. The aqueous phase was transferred into a new vesseland mixed with 1 ml isopropanol. For precipitating the RNA the samplewas stored at −20 C. for 1 hour. After renewed centrifugation at 4° C.at 10000 g the RNA was pelleted. The pellet was resuspended in 0.3 mlbuffer (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5%sarcosyl, 0.1 M 2-mercaptoisopropanol), transferred into a new 1.5 mlEppendorf vessel and mixed with 1 volume of isopropanol. Afterincubation at −20° C. for 1 hour the solution was centrifuged in anEppendorf centrifuge at 4° C. for 10 minutes. The RNA pellet wasreceived in 75% ethanol and concentrated by centrifugation (Speed vac)and dried. For further processing the sample was dissolved in 100 μl 10mM Tris-HCl, pH 6.5.

EXAMPLE 5

Importance of reducing reagents (such as DTT) in the stabilizingsolution for the longterm stability of RNA

Material and Method

Stabilizing solution used: 4.0 M GTC; 13.5% Triton X100; 45 mMTris//HCl; with or without 120 mM DTT. 700 μl serum was mixed with 700μl stabilizing solution. After incubation for 2 min 20 μl MS2-RNA (0.8μg/μl of Roche) was added. The samples were incubated at 40° C. for 180min and then processed in aliquots of 400 μl each with the High Puretotal RNA Kit of Roche. The samples were applied in one step to thecolumn without addition of the binding reagent of the kit andcentrifuged in accordance with the instructions. The following washsteps and the elution of the RNA in 50 μl elution buffer were carriedout in accordance with the instructions.

The analysis was carried out by means of agarose gel (see FIG. 6).Result: Without the addition of reducing reagents to the stabilizingsolution no long-term stabilization of RNA can be achieved.

EXAMPLE 6

Stability of Free MS2-RNA in Serum. Kinetics of the RNA Degradation bySample Components

Material and Method

250 μl serum was spiked with 10 μl MS2-RNA (0.8 μg/μl of Roche) andincubated at room temperature. Immediately after the addition of RNA,after 2 min to 50 min, the natural RNA degradation in serum was stoppedby adding 250 μl stabilizing solution. All batches were analyzed twice.As a standard, a sample was only mixed with MS2-RNA after addition ofthe stabilizing solution to the serum and was processed in parallel.

All samples were processed in parallel with the High Pure viral RNA Kitof Roche. The samples were applied to the column in one step withoutaddition of the binding reagent of the kit and centrifuged according toinstructions. The following wash steps and the elution of RNA in 50 μlelution buffer were carried out according to instructions. 20 μl of theeluate was separated by means of a 1.2% native agarose gel and analyzed(see FIG. 7).

Result: MS2-RNA is not stable in serum. Already 2 minutes after additionof RNA to the serum the RNA is completely degraded. By the addition ofstabilizing solution to the serum in the ratio of 1:1, this process canbe stopped immediately, and a stabilization of the RNA can be achievedat the time when the stabilizing solution is added (=blood withdrawal).

EXAMPLE 7

Stability of MS2-RNA in Serum/stabilization Solution. Dependence on theGTC Concentration

Material and Method

Stabilization solutions used: 3-5 M GTC; 13.5% Triton X100; 50 mM DTT;42 mM Tris/HCl

pH of the solutions: about 4.0

pH of the solutions after addition of serum: about 6.7.

2 ml serum was mixed with 2.5 ml of the respective stabilizationsolutions. After an incubation time of 2 to 5 min 90 μl MS2-RNA (0.8μg/μl of Roche) was added and incubated at 40° C. 400 μl samples weretaken at regular intervals and processed with the High Pure total RNAKit of Roche according to Example 5. The samples were eluted in 50 μland frozen at −20° C. For the analysis of the RNA integrity 20 μl of theeluate was applied to a 1.5% agarose gel (see FIG. 8).

For the PCR analysis of the samples 10 μl of the eluate was reverselytranscribed by means of AMV-RT (Roche) and subsequently analyzed bymeans of PCR on the Lightcycler:

Batch for RT: 4.0 μl AMV-RT buffer (42° C. for 1 h) 2.0 μl dNTP's (finalconcentration 10 mM) 0.5 μl RNase inhibitor (Roche, 20 units) 1.0 μlPrimer 2827 (final concentration 1 μM) 1.9 μl DMPC water 0.6 μl AMV-RT(Roche, 15 units) 10 μl template RNA      Σ 20 μl

The PCR was carried out on the Lightcycler at an annealing temperatureof 61° C. using SYBR-Green as detection system. Batch for PCR:

1.6 μl MgCl₂ (batch solution 25 mM) 5.9 μl DMPC water 0.25 μl Primer2827 (batch solution 20 mM) 0.25 μl Primer 2335 (batch solution 20 mM)1.0 μl SYBR-Green-Mastermix (Roche) 1.0 μl RT batch (1:50 diluted) Σ 10μ1

The amplificate of the PCR was completely applied to a 2% agarose gel(see FIG. 9).

Result:

RNA integrity at 40° C. after 3 days.

The agarose gel in FIG. 8 shows 20 μl of the eluted MS2-RNA afterincubation at 40° C. for 3 days. After this period distinct differencesin the RNA integrity can be made out in dependence upon the GTC content.Thus a salt content of less than 2 M in the serum/stabilization solutionis of advantage to the integrity of the RNA.

Amplificability of the RNA at 40° C. after 8 days.

Although a beginning degradation of the RNA was already detected at 40°C. after 3 days, all of the RNA samples could be amplified after anincubation of 8 days at 40° C. and clearly detected.

The amplificate of the PCR was fully applied to a 2% agarose gel (seeFIG. 9).

EXAMPLE 8

Stability of MS2-RNA in Serum/stabilization Solution: Dependence on thepH of the Sample Mixed with Stabilization Solution

Material and Method

Solution used: 4M (5M) GTC 14.4% Triton X 100 50 mM DTT 45 mM Tris/HClpH after serum addition between 6.7 and 8.0

2.5 ml stabilization solution was mixed with 2.0 ml serum. Afteraddition of 90 μl MS2-RNA (0.8 μg/μl, Roche) the samples were incubatedat room temperature. The RNA was processed at regular intervals from 500μl sample with the Roche viral RNA kit according to Example 6 andisolated in 50 μl elution buffer. 20 μl of the eluate was analyzed bymeans of agarose gel (see FIG. 10).

Result:

The pH of the serum/stabilization solution and thus the pH and thebuffer range of the stabilization solution are decisive for thelong-term stabilization of RNA. While at a pH of 8.0 an intact RNA couldno longer be detected already after 2 days, intact RNA is stilldetectable within a pH range between 6.6 and 7.0 after 13 days ofincubation at room temperature.

Apart from the pH, however, an optimally adjusted GTC concentration isalso of importance to the long-term stabilization of RNA (see alsoExample 7). The illustrated example demonstrates that a GTC finalconcentration in the stabilized sample of 2.2 M GTC is better than 2.78for a long-term stabilization of RNA.

Legends

FIG. 1:

Sampling vessel with N-sS, defined vacuum, sealed with septum.

FIG. 2:

Gel analysis (1% agarose) of RNA which was stored in the sampling vesselfor different periods of time. Column 1: Isolation directly aftersampling (no storage), column 2: storage for one month at −20° C.,column 3: storage for 6 days at 4° C. The amount of the applied RNAcorresponded to a blood volume of 120 μl.

FIG. 3:

Gel analysis (1% agarose) of DNA which was stored in the sampling vesselfor different periods of time. Column 1: isolation directly aftersampling (no storage), column 2: storage for one month at −20° C.,column 3: storage for 6 days at 4° C. The amount of the applied DNAcorresponded to a blood volume of 10 μl.

FIG. 4:

Gel analysis (1% agarose) of mRNA which was isolated from 10 ml blood(column 2). Molecular weight marker (column 1). In addition to the mRNA,the rRNA bands are visible. The sharp contours of the bands demonstratethe integrity of the nucleic acids.

FIG. 5:

Gel analysis (1% agarose) of the RNA which was isolated from 120 μlblood.

FIG. 6:

Gel analysis of isolated MS2-RNA after incubation in serum/stabilizationsolution with/without DTT for 180 min at 40° C.

Column 1: positive control: MS2-RNA, column 2: DNA marker, column 3,4,5:MS2-RNA after incubation with DTT-containing stabilization solution,column 6,7,8: MS2-RNA after incubation with stabilization solutionwithout DTT.

FIG. 7:

Gel analysis of isolated MS2-RNA after incubation in serum for 0-50 min

Column 10,17: MS2-RNA standard, column 9,16: DNA marker, column 7,8:incubation for 0 min, column 5,6: incubation for 2 min, column 3,4:incubation for 5 min, column 1,2: incubation for 10 min, column 11,12:incubation for 15 min, column 13,14: incubation for 30 min, column 15:incubation for 50 min

FIG. 8:

Gel analysis of MS2-RNA which was isolated after incubation inserum/stabilization solution for 3 days at 40° C. The GTC content of thestabilization solution after serum addition in which the relevant RNAsample was incubated is indicated in the corresponding column. Column 1:2.70 M GTC, column 2: 2.5 M GTC, column 3: 2.36 M GTC, column 4: 2.20 MGTC, column 5: 2.08 M GTC, column 6: 1.94 M GTC, column 7: 1.80 M GTC,column 8: 1.66 M GTC.

FIG. 9:

Gel analysis of the PCR amplificates of MS2-RNA which was isolated after1 day and 8 days, respectively, of incubation at 40° C. inserum/stabilization solution.

Column 1: Amplificate of the RNA isolated after 1 day, column 2:amplificate of the RNA isolated after 8 days, column 3: DNA marker,column 4: MS2-RNA positive control: 0.8 μg in 10 μl RT, 1:50 diluted, 1μl amplified.

FIG. 10:

Gel analysis of isolated MS2-RNA after 6 days (column 2-12) and 13 days(column 14-19), respectively, of incubation at room temperature inserum/stabilization solution. The pH which was obtained after mixing ofserum and stabilization solution is written behind the correspondingcolumns.

Column 1, 13, 20: DNA marker, column 2: pH 8.0, column 3: pH 7.7, column4: pH 7.5, column 5: pH 7.35, column 6: pH 7.18, column 7,14: pH 7.07,column 8,15: pH 6.94, column 9,16: pH 6.8, column 10,17: pH 6.72, column11,18: pH 6.68 and column 12,19: pH 6.7. The stabilization solution ofRNA in column 12, 19 had the same pH as that of the RNA in column 11,but contained 5 m GTC instead of 4 M.

1. A blood withdrawing vessel comprising: an evacuated chamber which isprovided for receiving withdrawn blood containing a nucleicacid-stabilizing aqueous solution for stabilizing nucleic acids in thewithdrawn blood directly upon contact with the solution, wherein thesolution comprisingcomprises the following components: a guanidiniumsalt in a concentration of 1 to 8.0 M; a buffer substance in aconcentration of 10 to 300 mM; a reducing agent in a concentration of0.1 to 10%, by wt; and a detergent in a concentration of 5 to 30%, bywt.
 2. The vessel according to claim 1, characterized in that theguanidinium salt is selected from guanidinium thiocyanate andguanidinium chloride.
 3. The vessel according to claim 1, characterizedin that the guanidinium salt is present in a concentration of 2.5 to 8.0M.
 4. The vessel according to claim 1, characterized in that the buffersubstance is selected from Tris, HEPES, MOPS, citrate and phosphatebuffer.
 5. The vessel according to claim 1, characterized in that thedetergent is selected from Triton-X-100, NP-40, polydocanol and Tween20.
 6. The vessel according to claim 1, characterized in that thereducing agent is selected from dithiothreitol, β-mercaptoethanol andTCEP.
 7. The vessel according to claim 1, characterized in that the pHof the solution is between 4.0 and 7.5.
 8. The vessel according to claim1, characterized in that the solution contains the following components:4 m guanidinium thiocyanate; 45 mM Tris/HCl; 15% (w/v) Triton-X-100;0.8% (w/v) DTT; wherein the PH pH is at 6.0.
 9. The vessel according toclaim 1, characterized in that it has a vacuum in the chamber which isprovided for receiving blood.
 10. The vessel according to claim 1,characterized in that it contains withdrawn blood.
 11. A method ofwithdrawing blood, comprising the step of directly introducing the bloodinto a vessel according to claim
 1. 12. The method according to claim11, characterized in that an amount of blood is withdrawn that is 0.1 to4 times the volume of the solution in the vessel.
 13. The methodaccording to claim 12, characterized in that the concentration of theguanidinium salt after the blood is introduced is between 1.0 M and 5 M.14. A method for stabilizing and/or isolating nucleic acids from blood,comprising the step of introducing blood into a vessel according toclaim 1 and, optionally, isolating the nucleic acids with conventionalmethods.
 15. The method according to claim 11, characterized in that thepH of the resultant mixture of the solution is adjusted such that,following the introduction of the blood, a pH between 4.0 and 7.5 isobtained and blood is between 5.0 and 7.6.
 16. The vessel according toclaim 7, characterized in that the pH of the solution is between 4.0 and6.5 5.0 and 6.0.
 17. The method according to claim 13, characterized inthat the concentration of the guanidinium salt, after blood isintroduced, is between 1.5 and 5 M.
 18. A blood withdrawing vesselcomprising: an evacuated chamber which is provided for receiving bloodcontaining a nucleic acid-stabilizing aqueous solution for stabilizingnucleic acids in the blood directly upon contact with the solution,wherein the solution comprisingcomprises the following components: aguanidinium salt in a concentration of 1 to 8.0 M; a buffer substance ina concentration of 10 to 300 mM; a reducing agent in a concentration of0.1 to 10%, by wt.
 19. The vessel according to claim 18, characterizedin that the guanidinium salt is selected from guanidinium thiocyanateand guanidinium chloride.
 20. The vessel according to claim 19,characterized in that the guanidinium salt is present at a concentrationof 2.5 to 8.0 M.
 21. The vessel according to claim 18, characterized inthat the buffer substance is selected from Tris, HEPES, MOPS, citrateand phosphate buffer.
 22. The vessel according to claim 18,characterized in that the reducing agent is selected fromdithiothreitol, β-mercaptoethanol and TCEP.
 23. The vessel according toclaim 18, characterized in that the pH of the solution is between 4.0and 7.5.
 24. The vessel according to claim 23, characterized in that thepH of the solution is between 4.0 5.0 and 6.5.
 25. The vessel accordingto claim 18, characterized in that it has a vacuum in the chamber whichis provided for receiving blood.
 26. The vessel according to claim 18,characterized in that it contains withdrawn blood.
 27. A method ofwithdrawing blood, comprising the step of directly introducing the bloodinto a vessel according to claim
 18. 28. The method according to claim27, characterized in that an amount of blood is withdrawn that is 0.1 to4 times the volume of the solution in the vessel.
 29. The methodaccording to claim 28, characterized in that the final concentration ofthe guanidinium salt after blood supply is between 1.0 M and 5 M. 30.The method according to claim 29, characterized in that the finalconcentration of the guanidinium salt after blood supply is between 1.5M and 5 M.
 31. The method according to claim 29, characterized in thatthe pH of the resultant mixture of the solution is adjusted such that,following the addition of the sample material, a pH between 4.0 and 7.5is obtained and blood is between 5.0 and 7.6.
 32. A method forstabilizing and/or isolating nucleic acids from blood, comprising thestep of introducing blood into a vessel according to claim 18 and,optionally, isolating the nucleic acids with conventional methods.
 33. Astabilized blood sample obtainable by introducing whole blood into avessel according to claim
 18. 34. The blood sample according to claim33, characterized in that it has a pH of 4.0 to 7.5 5.0 to 7.6.
 35. Theblood sample according to claim 34, characterized in that it has a pH of6.6 to 7.0 6.3 to 6.9.
 36. The blood sample according to claim 33,characterized in that it is derived from human blood.
 37. A bloodwithdrawing vessel comprising an evacuated chamber which is provided forreceiving blood containing a nucleic acid-stabilizing aqueous solutionfor stabilizing nucleic acids in the withdrawn blood directly uponcontact with the solution, wherein the solution comprisingcomprises thefollowing components: a guanidinium salt in a concentration of 1 to 8.0M; a buffer substance in a concentration of 10 to 300 mM; a detergent ina concentration of 5 to 30%, by wt.
 38. The vessel according to claim37, characterized in that the guanidinium salt is selected fromguanidinium thiocyanate and guanidinium chloride.
 39. The vesselaccording to claim 38, characterized in that the guanidinium salt ispresent at a concentration of 2.5 to 8.0 M.
 40. The vessel according toclaim 37, characterized in that the detergent is selected fromTriton-X-100, NP-40, polydocanol and Tween
 20. 41. The vessel accordingto claim 37, characterized in that the buffer substance is selected fromTris, HEPES, MOPS, citrate and phosphate buffer.
 42. The vesselaccording to claim 37, characterized in that the further comprising areducing agent, wherein said reducing agent is a material selected fromthe group consisting of dithiothreitol, β-mercaptoethanol and TCEP. 43.The vessel according to claim 37, characterized in that the pH of thesolution is between 4.0 and 7.5.
 44. The vessel according to claim 43,characterized in that the pH of the solution is between 4.0 5.0 and 6.5.45. The vessel according to claim 37, characterized in that it has avacuum in the chamber which is provided for receiving blood.
 46. Thevessel according to claim 37, characterized in that it containswithdrawn blood.
 47. A method of withdrawing blood, comprising the stepof directly introducing the blood into a vessel according to claim 37.48. The method according to claim 47, characterized in that an amount ofblood is withdrawn that is 0.1 to 4 times the volume of the solution inthe vessel.
 49. The method according to claim 48, characterized in thatthe final concentration of the guanidinium salt after blood supply isbetween 1.0 M and 5 M.
 50. The method according to claim 48,characterized in that the final concentration of the guanidinium saltafter blood supply is between 1.5 M and 5 M.
 51. The method according toclaim 37 47, characterized in that the pH of the resultant mixture ofsolution is adjusted such that, following the addition of the samplematerial, a pH between 4.0 and 7.5 is obtained and blood is between 5.0and 7.6.
 52. A method for stabilizing and/or isolating nucleic acidsfrom blood, comprising the step of introducing blood into a vesselaccording to claim 37 and, optionally, isolating the nucleic acids withconventional methods.
 53. A stabilized blood sample obtainable by introducing introducing whole blood into a vessel according to claim
 37. 54.The blood sample according to claim 53, characterized in that it has apH of 4.0 to 7.5 5.0 to 7.6.
 55. The blood sample according to claim 53,characterized in that it has a pH of 6.6 to 7.0 6.3 to 6.9.
 56. Theblood sample according to claim 53, characterized in that it is derivedfrom human blood.
 57. A process for collecting and stabilizing a bloodsample, the process comprising: providing a vessel comprising aninterior, the interior comprising an aqueous solution that comprises aguanidinium salt, a buffer, a reducing agent and a detergent; anddrawing the blood sample from a blood vessel directly into the interior,wherein the aqueous solution causes lysis of cells in the blood andstabilizes nucleic acids in the sample.
 58. The process of claim 57,wherein the interior is evacuated and wherein the evacuation is used todraw the blood sample.
 59. The process of claim 58, wherein the vesselcomprises a tube having an open end sealed by a septum.
 60. The processof claim 58, wherein the evacuation is effective for drawing a specificvolume of the blood into the interior.
 61. The process of claim 57,wherein the guanidinium salt, the buffer, the reducing agent and thedetergent are present in amounts effective to provide cell lysis andstabilization of nucleic acids in the sample by inactivation ofnucleases.
 62. The process of claim 57, wherein the buffer is effective,upon drawing the blood into the interior, to provide the resultantmixture with a pH of 5.0 to 7.6.
 63. The process of claim 62, whereinthe buffer is effective, upon drawing the blood into the interior, toprovide the resultant mixture with a pH of 6.3 to 6.9.
 64. The processof claim 60, wherein the specific volume is about 0.1 to about 4 timesthe volume of the solution.
 65. The process of claim 57, wherein theguanidinium salt is one or more salts selected from the group consistingof guanidinium thiocyanate and guanidinium chloride.
 66. The process ofclaim 57, wherein the buffer is selected from the group consisting of:Tris (tris(hydroxymethyl)aminomethane), HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(4-morpholinepropanesulfonic acid), MES (4-morpholineethanesufonicacid), citrate and phosphate buffer.
 67. The process of claim 57,wherein the reducing agent is selected from the group consisting of: DTT(dithiothreitol), β-mercaptoethanol and TCEP(tris(2-carboxyethyl)phosphine).
 68. The process of claim 57, whereinthe detergent is selected from the group consisting of: (polyethyleneglycol tert-octylphenyl ether), (polyethylene glycol 4-nonylphenylether), Polydocanol (dodecyl polyethylene glycol ether) and(polyethylene glycol sorbitan monolaurate).
 69. The process of claim 57,wherein the pH of the aqueous solution in the vessel prior to thedrawing step is 4.0 to 7.5.
 70. The process of claim 69, wherein the pHof the aqueous solution in the vessel prior to the drawing step is 5 to6.
 71. The process of claim 57, wherein the solution comprises a singlebuffer, a single reducing agent and a single detergent.
 72. The processof claim 57, wherein the aqueous solution consists essentially of theguanidinium salt, the buffer, the reducing agent and the detergent. 73.The process of claim 57, further comprising the step of isolating thenucleic acids after the drawing step.
 74. The process of claim 73,wherein the isolating step is performed at least 3 days after thedrawing step.
 75. The process of claim 74, wherein the isolating step isperformed at least 6 days after the drawing step.
 76. The process ofclaim 75, wherein the isolating step is performed at least 8 days afterthe drawing step.
 77. A vessel comprising an interior, the interiorcomprising an aqueous solution that comprises a guanidinium salt, abuffer, a reducing agent and a detergent, wherein the interior isevacuated, and wherein the aqueous solution is capable of lysing bloodcells and stabilizing nucleic acids.
 78. The vessel of claim 77, whereinthe vessel comprises a tube having an open end sealed by a septum. 79.The vessel of claim 77, wherein the evacuation is effective for drawinga specific volume of a fluid sample into the interior.
 80. The vessel ofclaim 77, wherein the guanidinium salt, the buffer, the reducing agentand the detergent are present in amounts effective to provide cell lysisand stabilization of nucleic acids in a fluid sample by inactivation ofnucleases.
 81. The vessel of claim 79, wherein the buffer is effective,upon mixing with the specific volume of a blood sample, to provide theresultant mixture with a pH of 5.0 to 7.6.
 82. The vessel of claim 81,wherein the buffer is effective to provide the resultant mixture with apH of 6.3 to 6.9.
 83. The vessel of claim 77, wherein the pH of theaqueous solution in the vessel is 4.0 to 7.5.
 84. The vessel of claim83, wherein the pH is 5 to
 6. 85. The vessel of claim 79, wherein thespecific volume is about 0.1 to about 4 times the volume of thesolution.
 86. The vessel of claim 77, wherein the guanidinium salt isone or more salts selected from the group consisting of guanidiniumthiocyanate and guanidinium chloride.
 87. The vessel of claim 77,wherein the buffer is selected from the group consisting of: Tris(tris(hydroxymethyl)aminomethane), HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(4-morpholinepropanesulfonic acid), MES (4-morpholineethanesufonicacid), citrate and phosphate buffer.
 88. The vessel of claim 77, whereinthe reducing agent is selected from the group consisting of: DTTdithiothreitol, β-mercaptoethanol and TCEP(tris(2-carboxyethyl)phosphine).
 89. The vessel of claim 77, wherein thedetergent is selected from the group consisting of: (polyethylene glycoltert-octylphenyl ether), (polyethylene glycol 4-nonylphenyl ether),Polydocanol (dodecyl polyethylene glycol ether) and (polyethylene glycolsorbitan monolaurate).
 90. The vessel of claim 77, wherein the solutioncomprises a single buffer, a single reducing agent and a singledetergent.
 91. The vessel of claim 77, wherein the aqueous solutionconsists essentially of the guanidinium salt, the buffer, the reducingagent and the detergent.
 92. A process for collecting and stabilizing ablood sample, the process comprising: providing a vessel comprising aninterior, the interior comprising an aqueous solution that comprises aguanidinium salt, a buffer and a reducing agent; and drawing the bloodsample from a blood vessel directly into the interior, wherein theaqueous solution causes lysis of cells in the blood and stabilizesnucleic acids in the sample.
 93. The process of claim 92, wherein theinterior is evacuated and wherein the evacuation is used to draw theblood sample.
 94. The process of claim 93, wherein the vessel comprisesa tube having an open end sealed by a septum.
 95. The process of claim93, wherein the evacuation is effective for drawing a specific volume ofthe blood into the interior.
 96. The process of claim 92, wherein theguanidinium salt, the buffer and the reducing agent are present inamounts effective to provide cell lysis and stabilization of nucleicacids in the sample by inactivation of nucleases.
 97. The process ofclaim 92, wherein the buffer is effective, upon drawing the blood intothe interior, to provide the resultant mixture with a pH of 5.0 to 7.6.98. The process of claim 97, wherein the buffer is effective, upondrawing the blood into the interior, to provide the resultant mixturewith a pH of 6.3 to 6.9.
 99. The process of claim 95, wherein thespecific volume is about 0.1 to about 4 times the volume of thesolution.
 100. The process of claim 92, wherein the guanidinium salt isone or more salts selected from the group consisting of guanidiniumthiocyanate and guanidinium chloride.
 101. The process of claim 92,wherein the buffer is selected from the group consisting of: Tris(tris(hydroxymethyl)aminomethane), HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(4-morpholinepropanesulfonic acid), MES (4-morpholineethanesufonicacid), citrate and phosphate buffer.
 102. The process of claim 92,wherein the reducing agent is selected from the group consisting of: DTT(dithiothreitol), β-mercaptoethanol and TCEP(tris(2-carboxyethyl)phosphine).
 103. The process of claim 92, whereinthe pH of the aqueous solution in the vessel prior to the drawing stepis 4.0 to 7.5.
 104. The process of claim 103, wherein the pH of theaqueous solution in the vessel prior to the drawing step is 5 to
 6. 105.The process of claim 92, further comprising the step of engaging thevessel with a blood sampling accessory.
 106. The process of claim 92,wherein the solution comprises a single buffer and a single reducingagent.
 107. The process of claim 92, wherein the aqueous solutionconsists essentially of the guanidinium salt, the buffer and thereducing agent.
 108. The process of claim 92, further comprising thestep of isolating the nucleic acids after the drawing step.
 109. Theprocess of claim 108, wherein the isolating step is performed at least 3days after the drawing step.
 110. The process of claim 109, wherein theisolating step is performed at least 6 days after the drawing step. 111.The process of claim 110, wherein the isolating step is performed atleast 8 days after the drawing step.
 112. A vessel comprising aninterior, the interior comprising an aqueous solution that comprises aguanidinium salt, a buffer, and a reducing agent, wherein the interioris evacuated, and wherein the aqueous solution is capable of lysingblood cells and stabilizing nucleic acids.
 113. The vessel of claim 112,wherein the vessel comprises a tube having an open end sealed by aseptum.
 114. The vessel of claim 112, wherein the evacuation iseffective for drawing a specific volume of a fluid sample into theinterior.
 115. The vessel of claim 112 wherein the guanidinium salt, thebuffer and the reducing agent are present in amounts effective toprovide cell lysis and stabilization of nucleic acids in a fluid sampleby inactivation of nucleases.
 116. The vessel of claim 114, wherein thebuffer is effective, upon mixing with the specific volume of a bloodsample, to provide the resultant mixture with a pH of 5.0 to 7.6. 117.The vessel of claim 116, wherein the buffer is effective to provide theresultant mixture with a pH of 6.3 to 6.9.
 118. The vessel of claim 112,wherein the pH of the aqueous solution in the vessel is 4.0 to 7.5. 119.The vessel of claim 118, wherein the pH is 5 to
 6. 120. The vessel ofclaim 114, wherein the specific volume is about 0.1 to about 4 times thevolume of the solution.
 121. The vessel of claim 112, wherein theguanidinium salt is one or more salts selected from the group consistingof guanidinium thiocyanate and guanidinium chloride.
 122. The vessel ofclaim 112, wherein the buffer is selected from the group consisting of:Tris (tris(hydroxymethyl)aminomethane), HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(4-morpholinepropanesulfonic acid), MES (4-morpholineethanesufonicacid), citrate and phosphate buffer.
 123. The vessel of claim 112,wherein the reducing agent is selected from the group consisting of: DTT(dithiothreitol), β-mercaptoethanol and TCEP(tris(2-carboxyethyl)phosphine).
 124. The vessel of claim 112, whereinthe solution comprises a single buffer and a single reducing agent. 125.The vessel of claim 112, wherein the aqueous solution consistsessentially of the guanidinium salt, the buffer and the reducing agent.126. A process for collecting and stabilizing a blood sample, theprocess comprising: providing a vessel comprising an interior, theinterior comprising an aqueous solution that comprises a guanidiniumsalt, a buffer and a detergent; and drawing the blood sample from ablood vessel directly into the interior, wherein the aqueous solutioncauses lysis of cells in the blood and stabilizes nucleic acids in thesample.
 127. The process of claim 126, wherein the interior is evacuatedand wherein the evacuation is used to draw the blood sample.
 128. Theprocess of claim 127, wherein the vessel comprises a tube having an openend sealed by a septum.
 129. The process of claim 127, wherein theevacuation is effective for drawing a specific volume of the blood intothe vessel.
 130. The process of claim 126, wherein the guanidinium salt,the buffer and the detergent are present in amounts effective to providecell lysis and stabilization of nucleic acids in the sample byinactivation of nucleases.
 131. The process of claim 126, wherein thebuffer is effective, upon drawing the blood into the interior, toprovide the resultant mixture with a pH of 5.0 to 7.6.
 132. The processof claim 131, wherein the buffer is effective, upon drawing the bloodinto the interior, to provide the resultant mixture with a pH of 6.3 to6.9.
 133. The process of claim 129, wherein the specific volume is about0.1 to about 4 times the volume of the solution.
 134. The process ofclaim 126, wherein the guanidinium salt is one or more salts selectedfrom the group consisting of guanidinium thiocyanate and guanidiniumchloride.
 135. The process of claim 126, wherein the buffer is selectedfrom the group consisting of: Tris (tris(hydroxymethyl)aminomethane),HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(4-morpholinepropanesulfonic acid), MES (4-morpholineethanesufonicacid), citrate and phosphate buffer.
 136. The process of claim 126,wherein the one detergent is selected from the group consisting of:(polyethylene glycol tert-octylphenyl ether), (polyethylene glycol4-nonylphenyl ether), Polydocanol (dodecyl polyethylene glycol ether)and (polyethylene glycol sorbitan monolaurate).
 137. The process ofclaim 126, wherein the pH of the aqueous solution in the vessel prior tothe drawing step is 4.0 to 7.5.
 138. The process of claim 137 whereinthe pH of the aqueous solution in the vessel prior to the drawing stepis 5 to
 6. 139. The process of claim 126, wherein the solution comprisesa single buffer and a single detergent.
 140. The process of claim 126,wherein the aqueous solution consists essentially of the guanidiniumsalt, the buffer and the detergent.
 141. The process of claim 126,further comprising the step of isolating the nucleic acids after thedrawing step.
 142. The process of claim 141, wherein the isolating stepis performed at least 3 days after the drawing step.
 143. The process ofclaim 142, wherein the isolating step is performed at least 6 days afterthe drawing step.
 144. The process of claim 143, wherein the isolatingstep is performed at least 8 days after the drawing step.
 145. A vesselcomprising an interior, the interior comprising an aqueous solution thatcomprises a guanidinium salt, a buffer and a detergent, wherein theinterior is evacuated, and wherein the aqueous solution is capable oflysing blood cells and stabilizing nucleic acids.
 146. The vessel ofclaim 145, wherein the vessel comprises a tube having an open end sealedby a septum.
 147. The vessel of claim 145, wherein the evacuation iseffective for drawing a specific volume of a fluid sample into theinterior.
 148. The vessel of claim 145, wherein the guanidinium salt,the buffer and the detergent are present in amounts effective to providecell lysis and stabilization of nucleic acids in a fluid sample byinactivation of nucleases.
 149. The vessel of claim 147, wherein thebuffer is effective, upon mixing with the specific volume of a bloodsample, to provide the resultant mixture with a pH of 5.0 to 7.6. 150.The vessel of claim 149, wherein the buffer is effective to provide theresultant mixture with a pH of 6.3 to 6.9.
 151. The vessel of claim 145,wherein the pH of the aqueous solution in the vessel is 4.0 to 7.5. 152.The vessel of claim 151, wherein the pH is 5 to
 6. 153. The vessel ofclaim 147, wherein the specific volume is about 0.1 to about 4 times thevolume of the solution.
 154. The vessel of claim 145, wherein theguanidinium salt is one or more salts selected from the group consistingof guanidinium thiocyanate and guanidinium chloride.
 155. The vessel ofclaim 145, wherein the buffer is selected from the group consisting of:Tris (tris(hydroxymethyl)aminomethane), HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(4-morpholinepropanesulfonic acid), MES (4-morpholineethanesufonicacid), citrate and phosphate buffer.
 156. The vessel of claim 145,wherein the detergent is selected from the group consisting of:(polyethylene glycol tert-octylphenyl ether), (polyethylene glycol4-nonylphenyl ether), Polydocanol (dodecyl polyethylene glycol ether)and (polyethylene glycol sorbitan monolaurate).
 157. The vessel of claim145, wherein the solution comprises a single buffer and a singledetergent.
 158. The vessel of claim 145, wherein the aqueous solutionconsists essentially of the guanidinium salt, the buffer and thedetergent.